The T-cell repertoire is generated through a tightly regulated developmental program of selection in which thymocytes bearing immunologically desirable T-cell receptor (TCR) specificities are preserved and those expressing harmful specificities are eliminated via an apoptotic mechanism termed negative selection Using representational difference analysis, we have cloned a novel inhibitor of NF-KB, IkBNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death The predicted protein contains 7 ankyrin repeats and is homologous to known IkB family members but lacks ubiquitination-based degradation signals In Class I and Class II MHC-restricted TCR transgenic mice, transcription of IkBNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides IkBNS blocks transcription from NF-kappaB reporters, alters NF-kappaB electrophoretic mobility shifts and interacts with NF-KappaB proteins in thymic nuclear lysates following TCR stimulation. Using an anti-IkBNS mAb, the kinetics of IkappaBNS protein expression in the thymus have been determined and are very different flora those of IkappaBalpha. In addition, IkappaBNS localizes to the nucleus. Over-expression of IkappaBNS using retroviral transduction in fetal thymic organ culture alters the development of thymocytes and enhances anti-CD3epsilon-induced cell death. In this proposal, I describe plans to further characterize this thymic NF-kappaB inhibitor. The effect of altered peptide ligands on IkappaBNS mRNA induction will be analyzed. The effects of IkappaBNS on negative selection in vivo will be analyzed in fetal thymic organ culture using mutant and wild type IkappaBNS, in transgenic mice expressing tissue specific IkBNS as well as in IkBNS gene deleted animals. I will also determine what proteins associate with IkBNS (NF-kB dimers or other proteins,). These studies will elucidate the role of IkBNS in the process of negative selection during thymic development. An understanding of the mechanism of negative selection may form the basis for manipulating the immune response allowing controlled elimination of autoreactive T cells and enhanced generation of T cells with medically relevant specificities.